Production of fungal extracellular immune stimulating compounds

ABSTRACT

A process is described for the production of an immunostimulant by submerged cultivation of  Lentinus edodes  in which mycelium from agar plates or a fermentation broth is added to a liquid medium in a shake flask or a bioreactor containing nutrients such as malt extract, yeast extract, peptone and glucose having access to air or to which air is added, and which is kept in constant movement at approx. 28° C. At the proper conditions, there will be an increase in the production of extracellular lentinan, which is shown to be a better immunostimulant than intracellular lentinan. The extracellular product is precipitated from the growth medium by means of methods for the precipitation of microbial polysaccharide.

TECHNICAL AREA OF THE INVENTION

The present invention is directed to a method for producing an immunestimulant in liquid culture. The invention is in one embodiment directedto culturing Lentinus edodes under particular conditions in afermentation broth. This application is a non-provisional claimingpriority from Norwegian patent application No. NO 2001 4256 filed 3 Sep.2001, which is hereby incorporated by reference in its entirety. Allpatent and non-patent references cited in that application, or in thepresent application, are also hereby incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

It is known that health-promoting effects are attributed to glucans fromfungi and yeasts. “Shiitake” fungus (Lentinus edodes) has beenattributed effects which can be exploited for many medicinal purposessuch as immunestimulation, anti-virus, anti-tumour, etc. Studies oflentinan have shown that it stimulates the immune system of the host ina variety of ways, such as activation of T helper cells, increasedproduction of Interleukin 1 and Interleukin 2, increased antibodyproduction in various forms of cancer, and decreasing the cholesterollevel in the blood. (Herbs for Health, jan/feb, 1997; K. Jones,“Shiitake: Medicine in a mushroom”, p. 40-50, 54; Anticancer Res, Vol.17(4A), 1997; H. Matsouka, “Lentinan potentiates immunity and prolongsthe survival time of some patients”, p. 2751-2755; Adv Appl Microbiol,Vol. 39, 1993; S. C. Jong, “Medicinal and therapeutic value of theshiitake mushroom”, p. 153-184, Int J Immunopharmacol, Vol. 14, 1992; K.Irinoda, “Stimulation of microbiocidal host defence mechanism againstaerosol influenza virus infection by lentinan, p. 971-977., Jpn J CancerRes, Vol. 76(1), 1985; D. Herlyn, “Monoclonal antibody-dependent murinemacrophage-mediated cytotoxicity against human tumors is stimulated bylentinan, p. 37-42).

One active ingredient of Lentinus edodes is termed lentinan, apolysaccharide based compound described as a beta-(1,3) glucan backbonewith beta-(1,6) side chains.

“Solid-state” reactors are routinely used for culturing fungi such asLentinus edodes. This is a technology which is used for many purposessuch as composting, production of biological products such as enzymes,soy sauce, acetic acid, and the like.

For the production of lentinan, Lentinus edodes can be cultivated on asuitable solid matrix provided by stems of tree or chips of wood towhich is often added chemical compounds supporting the growth ofmycelium and development of the fruiting bodies, where most of thelentinan is localised. The fruiting bodies are harvested, either by handor mechanically, and are subsequently dried and ground to a powder whichcan be used as it is, or used in tablets, or sent for further processingsuch as extraction of lentinan.

The methods used for culturing fungi such as Lentinus edodes, harvestingand subsequently drying the fruiting bodies, and optionally extractionof lentinan, are well known for the skilled person. The cultivation timecan be from four to ten weeks with few possibilities for controlling theprocess. This results in a fungal growth, and in turn, in an amount oflentinan produced which is not the same for each batch carried out.Also, extraction of lentinan from the fungal material is time consumingand process intensive.

Kim et al. (2001, Biotechnology Letters, 23, 513-517) describesextracellular polysaccharides produced by Phellinus lintus in submergedculture; Kim et al. (2002: Letters in Applied Microbiology, 34, 56-61)describes mycelial growth and exobiopolymer production by submergedculture of various edible mushrooms. There is no data available toindicate that any of these extracellular polymers are immunestimulating.

It is generally known to grow mycelium of Basidiomycetes in submergedculture and to obtain active ingredients from the growth medium, seee.g. Aouadi et al 1992 (Carbohydr Polym 17:177-184) and Lee et al 1995(Prog Plant Polym Carbohydr Res, B. Behrs Verlag, Hamburg DE) andHatvani 2001 (Int J Antimicrob Agents 17:71-). However, none of thereferences discloses the isolation of extracellular components withimmune stimulating activities. Furthermore, none of the referencesdisclose that extracellular polysaccharides have different activitiesfrom their intracellular counterparts.

It is therefore an object of the present invention to provide novelmethods for production of extracellular immune stimulating agents withhigher level of activity than the hitherto known intracellularcounterparts.

SUMMARY OF THE INVENTION

The invention in a first aspect, relates to a method for cultivating afungal mycelium, preferably a fungus from the class of basidiomycetes,such as a fungus of the genus Lentinus, such as Lentinus edodes, in aliquid medium of sterile water to which nutrient compounds are added inpredetermined concentrations. The liquid medium supports fungal growthand stimulates the production of extracellular compounds, such as immunestimulating agents, such as lentinan produced by fungal mycelium of thegenus Lentinus, such as e.g. Lentinus edodes.

By fungal mycelium is intended any fungal biomass, which can be grown insubmerged culture according to the invention. The fungal biomass may bein the form of single hyphae, spores, aggregates of mycelium, and partlydifferentiated mycelium.

Lentinan as used herein refers to the polysaccharide, which can beisolated from Lentinus edodes (Berk.) Sing. The primary structure is aβ-1,3-D-glucan having 2β-1,6-glucopyranoside branchings for every 5β-1,3 linear linkages. The molecular weight may vary from 400,000 to800,000 (Merck Index 12^(th) Edition, 1996, Monograph No 5462).

The cultivation of the fungal mycelium, such as fungi of the genusLentinus, such as Lentinus results in the production of one or moreextracellular agents, such as e.g. immune stimulating agents, in theform of e.g. a polysaccharide, such as e.g. a beta-glucan, includinglentinan isolatable from Lentinus edodes, a polypeptide, a glycosylatedpolypeptide or a proteinacious polysaccharide compound, a proteoglucan,such as e.g. a polypeptide associated alpha-glucan or a polypeptideassociated alpha-mannan, including KS-2 isolatable from Lentinus edodes,a lipid, or a secondary metabolite, which can be isolated and/orpurified, and optionally fractionated, from the extracellular fractionof the fermentation broth of a bioreactor following cultivation of thebasidiomycete fungal mycelium in question.

The extracellular fraction of the liquid fermentation medium is alsotermed the supernatant and this fraction can be separated from thefungal mycelium by e.g. centrifugation or filtration, or indeed by anyother means available for obtaining a liquid fraction essentiallywithout any fungal mycelium present therein. The term “essentiallywithout any fungal mycelium present therein” shall denote that theconcentration of fungal mycelium, including fractions thereof, has beenreduced at least by a factor of 10³, such as reduced by a factor of atleast 10⁴, for example a factor of at least 10⁵, such as reduced by afactor of at least 10⁶.

Besides being easier to isolate and process, fungal extracellular agentssuch as immune stimulating agents according to the present invention aresurprisingly more potent than fungal associated agents, i.e. agentsbeing either fungal intracellular agents, or agents which are removedfrom a liquid fermentation broth along with the removal of the fungalmycelium and fractions thereof by filtration, precipitation, orotherwise.

In a further aspect the invention relates to an immune stimulating agentobtainable from the extracellular part of the liquid growth mediumaccording to the method of producing said immune stimulating agent.

This immune stimulating agent has surprisingly turned out to have ahigher immunostimulating activity than the corresponding extractobtainable from the mycelium of the fungus, although the mycelium isknown as the primary source of e.g. lentinan. In a particularlypreferred embodiment, this extract is obtainable by removal of mycelium,removal of water from the medium, one or more rounds of precipitationwith alcohol and washing with acid and/or base.

According to the invention there is also provided a compositioncomprising the immune stimulating agent obtainable as described aboveand a physiologically acceptable carrier. Furthermore, there is provideda pharmaceutical composition comprising the immune stimulating agentaccording to the invention and a pharmaceutically acceptable carrier.The pharmaceutical composition may in particular be used for treatmentor prophylaxis in connection with an immune compromised condition.

Fungi belonging to the genus of Lentinus, such as Lentinus , edodesrepresent one example of fungi according to the present invention. Inother preferred aspects of the invention a method is provided whereinthe fungus is selected from the group of fungi consisting of Auriculariaauricula-judae, Coriolus versicolor, Grifola frondosa, Flammulinavelutipes, Schizophyllum commune, Sclerotinia scleroticum, Trametesversicolor, Tremella fuciformis, Agaricus blazei, Cordyceps sinensis,Ganoderma lucidum, Hericium erinaceus, lonotus obliquus, Pleurotusostreatus, and Polyperus umbellatus.

DETAILED DESCRIPTION OF THE INVENTION

For the method of the present invention, “liquid-state” cultivation ofLentinus mycelium, or fractions thereof, is used. In short, thisinvolves dissolving in water the nutrient compounds a microbial organismsuch as fungal mycelium, or fractions thereof, require for growth,transferring the solution to a bioreactor and inoculating the bioreactorwith cells or spores of the microbial organism such as a fungalmycelium, or fractions thereof, to be cultivated. This is done understerile conditions and with control of the environment in order to givethe microbial organism a suitable chemical and physical environment. Thetechnology related to “liquid-state” cultivation of microbial organismsis well known for the skilled person.

The advantage of “liquid-state” as compared to “semi-solid-state” or“solid-state” cultivation is that “liquid-state” proceeds more rapidly,is more effective with respect to conversion of the raw materials toproducts, and most importantly, it offers greater possibilities forcontrolling the growth conditions and thereby the reproducibility andthe predictability of the process.

What distinguishes the various “liquid-state” processes from one anotheris the microbial organisms which are used and the cultivationconditions. For production of lentinan the microbial organism will beLentinus edodes which is cultivated under the conditions describedbelow. During “liquid-state” culture the medium with the fungal biomassis preferably agitated to reduce the occurrence of gradients and toensure oxygen availability to the submerged cells. When microbialorganisms are grown in a bioreactor, oxygen may be supplied to theliquid medium and the level of dissolved oxygen may be controlled byknown methods.

Provided in one preferred embodiment is a method for production oflentinan, characterised by cultivating mycelium from Lentinus edodes ina liquid growth medium comprising one or more typical ingredientsrequired for growth of microbial organisms such as malt extract, yeastextract, peptone, glucose, sucrose, salts providing phosphate, magnesiumand potassium, corn-steep liquor and vitamins such as thiamine. Morepreferably, the medium comprises malt extract, yeast extract, peptoneand glucose for mycelium growth and production of lentinan.

For inoculation of the growth medium, Lentinus edodes mycelium from agarplates containing malt extract, yeast extract, peptone and glucose canbe used. Lentinus edodes can initially be cultivated on agar platescomprising the above nutrient compounds supporting the growth of thefungus. The plates are inoculated with mycelium from Lentinus edodes andincubated at least until a visible growth is evident on the plates, thiscan take from about 7 days to about 24 days or from about 10 to 30 days,typically 14 days or up to 20 days, at a temperature in the range offrom 18 to 32° C., preferably in the area of from 22 to 31° C., such asa temperature of about 23° C., for example 24° C., such as 25° C., forexample 26° C., such as 27° C., for example 28° C., such as 29° C., forexample 30° C. The temperature may also be from 18 to 37° C., preferablyfrom 23 to 32° C. such as about 28° C.

As an alternative to inoculation with mycelium from agar plates,inoculation of the growth medium can be carried out by using Lentinusedodes mycelium from a fermentation broth in e.g. a shake flask mediumcomprising nutrient compounds supporting cell growth. Shake flasks forcultivating Lentinus edodes can initially be inoculated with themycelium which is cultivated on agar plates. The mycelium is scraped offthe plates and transferred aseptically to shake flasks containingsterile water comprising dissolved nutrient compounds and nutrient saltssupporting the growth of the fungal mycelium. A typical growth mediumcontains glucose, peptone, yeast extract and malt extract. The amount ofinoculation material which gives the highest production of extracellularlentinan can be selected following initial experiments.

The shake flasks can be incubated by shaking for 6 to 21 days,preferably from 7 to 18 days, more preferably from 8 to 14 days at atemperature in the range of from 18 to 32° C., preferably in the area offrom 22 to 31° C., such as a temperature of about 23° C., for example24° C., such as 25° C., for example 26° C., such as 27° C., for example28° C., such as 29° C., for example 30° C. The shake flasks may also beincubated from 8-25 days, more preferably from 10-20 days, morepreferably from 12-18 days. The temperature may also be from 18 to 37°C., preferably from 23 to 32° C. such as about 28° C.

The content of the shake flasks can be used for inoculating abioreactor. In that case, the reactor comprises a sterile solution ofnutrient compounds and nutrient salts in water for mono-culturecultivation of basidiomycete fungal mycelium, or fractions thereof, suchas Lentinus fungal mycelium, such as Lentinus edodes.

The bioreactor fermentation period is typically in the range of from 50hours to 300 hours, preferably in the range of from 80 hours to 270hours, and the temperature is kept constant in the range of 18 to 32°C., preferably in the area of from 22 to 31° C., such as a temperatureof about 23° C., for example 24° C., such as 25° C., for example 26° C.,such as 27° C., for example 28° C., such as 29° C., for example 30° C.The temperature may also be from 18 to 37° C., preferably from 23 to 32°C. such as about 28° C.

The reactor is fitted with an inlet for supplying air to thefermentation broth, and the fermentation broth is preferably kept undercontinuous agitation either as a result of the addition of air, or bymeans of a mixer device suitable for providing a good mixing of thecontent of the reactor.

It is preferred to adjust the pH of the growth medium to from about 3 toabout 7, such as a pH of from about 4.5 to about 6.5, for example a pHof about 6, before the growth medium is inoculated with fungal mycelium,or fractions thereof, such as L. edodes mycelium. After the initialadjustment, pH may be dropped naturally during the course of thefermentation, or controlled at a particular value in the range pH 3 to7, using addition of suitable pH-control agents, such as acid and base.The temperature of the growth medium is preferably in the range of from18 to 32° C., preferably in the area of from 22 to 31° C., such as atemperature of about 23° C., for example 24° C., such as 25° C., forexample 26° C., such as 27° C., for example 28° C., such as 29° C., forexample 30° C. The temperature may also be from 18 to 37° C., preferablyfrom 23 to 32° C. such as about 28° C.

Samples can be obtained from the bioreactor and analysed for biomass,metabolic products and nutrient compounds, the determinations of whichcan assist the operator of the bioreactor in the running of thefermentation process. Typical analyses routinely carried out aredetermination of biomass, residual sugar concentration and extracellularagent concentration, such as lentinan concentration in the case ofLentinus including Lentinus edodes. A person skilled in the art knowsthe methods for analysis which can be employed in this respect.

In the case of cultivation of Lentinus edodes, extracellular lentinanmay be removed from the liquid growth medium by precipitation with e.g.alcohol, or by other means which result in the isolation and/orpurification of microbial polysaccharides. It is an important aspect ofthe present invention that such isolation from the growth medium isperformed with gentle methods and is generally carried out attemperatures around room temperature. Therefore the extracted compoundsare not degraded or converted as may often be the case when using harshextraction conditions for extracting compounds from the mycelium or fromthe fruiting bodies of Lentinus edodes.

Lentinus edodes deposited under IHEM 18992 with the Belgian CoordinatedCollections of Microorganism (BCCM), 14 Rue J. Wytsman, B-1050Bruxelles, Belgium, represents one preferred strain of Lentinus edodes.Further strains of Lentinus edodes are available from culturecollections such as ATCC (American Type Culture Collection, P.O. Box1549, Manassas, Va. 20108, USA), CBC (Centraalbureau voorSchimmelcultures, PO Box 85167, 3508 AD Utrecht, THE NETHERLANDS) andDSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH,Mascheroder Weg 1b, 38124 Braunschweig GERMANY).

Additionally relevant Lentinus species, besides Lentinus edodes,includes Lentinus species such as: Lentinus albovelutinus G. Stev.(1964) =Rhodocybe albovelutina (G. Stev.) E. Horak (1971); Lentinusanthocephalus (Lév.) Pegler; Lentinus badius Bres.; Lentinus castoreusFr. (1838) =Lentinellus ursinus (Fr.) Kühner (1926); Lentinuschrysopeplus Berk. & M. A. Curtis (1869) =Cyptotrama asprata (Berk.)Redhead & Ginns (1980); Lentinus cochleatus Fr.; Lentinus concinnusPat.; Lentinus delicatus G. Stev. (1964) =Marasmius delicatus (G. Stev.)E. Horak (1971); Lentinus fasciatus Berk.; Lentinus hepatotrichus Berk.(1859) =Lentinellus ursinus (Fr.) Kühner (1926); Lentinus hyracinusKalchbr. (1880) =Lentinellus ursinus (Fr.) Kühner (1926); Lentinuslepideus sensu Colenso (1891); Lentinus lepideus (Fr.) Fr. (1825)=Lentinus suffrutescens (Brot.) Fr. (1825); Lentinus novaezelandiaeBerk. (1855) =Lentinellus ursinus (Fr.) Kühner (1926); Lentinuspulvinulus Berk. (1859) =Lentinellus pulvinulus (Berk.) Pegler (1965);Lentinus punctaticeps Berk. & Broome (1883); Lentinus punctaticeps cf.sensu Petersen, Nicholl & Hughes (1997); Lentinus pygmaeus Colenso(1887) =Lentinus zelandicus Sacc. & Cub. (1887); Lentinus sajor-caju(Fr.) Fr.; Lentinus squarrulosus Mont.; Lentinus strigosus (Schwein.)Fr. (1825); Lentinus suffrutescens (Brot.) Fr. (1825); and Lentinustuber-regium Fr.; Lentinus zelandicus Sacc. & Cub. (1887) (Ref:http://nzfungi.landcareresearch.co.nz).

Fungal Mycelium Extracellular Immunostimulants

Provided in one embodiment of the invention is an immune stimulatingagent, preferably a polysaccharide or a glycosylated polypeptide,obtained from the cultivation of basidiomycete fungal mycelium such asLentinus mycelium according to the method of the invention.

Provided in another embodiment of the invention is the use of anextracellular agent such as lentinan in a method for stimulating theimmune system of an individual in need of such stimulation. The lentinanis administered e.g. orally or subcutaneously to the individual in apharmaceutically effective amount capable of stimulating the immunesystem of the individual. The stimulation of the immune system can bedemonstrated by e.g. increased antibody production, by activation ofhelper T-cells, or by increased production of interleukins such asInterleukin 1 and Interleukin 2.

Provided in yet another embodiment of the invention is the use of anextracellular agent such as lentinan in the manufacture of a medicamentfor treating an immune compromised condition in an individual in need ofsuch treatment. An immune compromised condition in an individual isdemonstrated e.g. by an insufficient amount of antibodies, or adecreased antibody production, by an insufficient amount of helperT-cells, or a decreased production of helper T-cells in the individual,or by an insufficient amount of interleukins such as Interleukin 1 andInterleukin 2, or a decreased production of interleukins such asInterleukin 1 and Interleukin 2 in the individual.

“Insufficient amount” and “decreased production” as used herein aboveshall denote such amounts and productions which a medical expertconsiders as being below a predetermined level or value normallyassociated with a healthy individual. The amount and/or production willgenerally depend on factors such as age, general physical condition, andthe like. For this reason a predetermined level or value shall bedetermined on an individual basis by a medical expert. One indication ofan immune compromised condition in an individual is a graduallydecreasing number of antibodies, a gradually decreasing number of CD4(positive) cells, or a gradually decreasing number of T-helper cells perunit (blood) sample volume measured over time, such as days, weeks,months or years.

Additional examples of extracellular immunostimulants includes, but isnot limited to, β-(1,3), β-(1,6) D-glucans, schizophyllan, grifolan,coriolan and Coriolus versicolor glycosylated polypeptides such as PSKand PSP, polypeptides associated with alpha-mannan such as KS-2isolatable from Lentinus edodes, and reishi isolatable from Ganodermalucidum. Reference is made to Table 1 herein below for a furtherdetailed description of the structure and composition of the aboveextracellular immunostimulants.

Preferred immunostimulating agents produced by fungal mycelium of theinvention are polysaccharides such as polysaccharides comprising aβ-D-glucan backbone (i.e. linear polymers of D-glucose with othermonosaccharides), or β-D-glucans linked to proteins (so-calledpolysaccharide-peptides, or “proteoglucans”). The preferredpolysaccharides are homopolymers. The basic β-D-glucan is a repeatingstructure with its D-glucose molecule joined by linear chains by β-bondsfrom the carbon 1 of one saccharide ring to the carbon 3 of the next(β-1,3), from carbon 1 to carbon 4 (β-1,4), or from carbon 1 to carbon 6(β-1,6). The best know immune stimulating compound produced by Lentinusedodes is lentinan. This is β-(1,3) D-glucan with β-(1,6) side chains.The chain length varies with the typical product having a molecularweight in the range 400,000 -1,000,000 g/mol. Further preferredpolysaccharides are listed in Table 1.

For a determination of the immunostimulating characteristics of anextracellular agent such as e.g. lentinan, the following method can beused: 12 weeks old Sprague Dawley rats receive 1 mg of test compoundwhich have been extracted from the fermentation broth in 0.5 ml 0.09saline (i.p.) 2 days before the immunisation. Control animals receive 1mg casein. The animals are immunised with BSA (0.5 mg) in 0.25 “FreundsComplete Adjuvant” and blood samples are obtained after 11 days formeasurement of the antibody response. The specific anti-BSA antibodyconcentration is determined against an absolute standard of antibody BSAby means of “sandwich” ELISA. A typical result of the immuno-stimulatingactivity of intracellular and extracellular lentinan is shown in Table7a and 7b.

Additional Fungal Mycelium Extracellular Agents and Uses Thereof

Further additional preferred embodiments of the present invention relateto extracellular agents, other than immunostimulating agents, producedby a number of fungi, as well as to methods for producing said agentsand methods for using said agents.

The fungi of particular interest to the present invention includeLentinus edodes, Ganoderma lucidum, Auricularia auricula-judae, Coriolusversicolor, Grifola frondosa, Flammulina velutipes, Schizophyllumcommune, Sclerotinia scleroticum, Trametes versicolor, Tremellafuciformis, Agaricus blazei, Cordyceps sinensis, Ganoderma lucidum,Hericium erinaceus, lonotus obliquus, Pleurotus ostreatus, and Polyperusumbellatus.

The above fungal mycelium can be cultivated in mono-culture in abio-reactor under suitable growth conditions allowing growth andpropagation of the fungal mycelium in question. The growth conditionscited herein for Lentinus edodes can also be used for the above fungalmycelium.

Besides extracellular fungal immune stimulating agents, such asextracellular proteinacious compounds, including glycosylatedpolypolypeptides, such as the alpha-mannan polypeptide KS-2, andextracellular polysaccharides, such as extracellular lentinan producedby Lentinus edodes, the fungal microbial organisms of the invention,such as e.g. Ganoderma lucidum, Auricularia auricula-judae, Coriolusversicolor, Grifola frondosa, Flammulina velutipes, Schizophyllumcommune, Sclerotinia scleroticum, Trametes versicolor, Tremellafuciformis, Agaricus blazei, Cordyceps sinensis, Ganoderma lucidum,Hericium erinaceus, lonotus obliquus, Pleurotus ostreatus, and Polyperusumbellatus are also capable of extracellularly producing agents havingadditional desirable effects.

TABLE 1 Extracellular agents, including structure and composition,produced by the selected fungi. Extracellular Fungus agent Structure andcomposition Schizophyllum commune schizophyllan β-(1,3), β-(1,6) Dglucan Grifola frondosa grifolan β-(1,3), β-(1,6) D glucan Coriolusversicolor PSK, PSP Polypeptides attached to polysaccaride β-D glucanchains. The polysaccharide chains are true glucans, mainly 1,4; 1,2 and1,3 glucose linkages Lentinus edodes KS-2 Polypeptide associatedα-mannan Lentinus edodes lentinan β-(1,3), β-(1,6) D glucan Ganodermalucidum reishi The basic structure of the major bioactive Ganodermaglucans β-(1,3), β-(1,6) D glucan is β-(1,3) D-glucopyronan with 1-5units of β-(1,6) monoglucosyl side chains

The effects associated with the above extracellular agents produced bythe fungi of the invention are e.g. analgesic effects, anti-allergicactivity, bronchitis-preventative effects, anti-inflammatory activity,anti-bacterial properties (against Staphylococci, Streptococci andBacillum pneumoniae), antioxidant effects, antitumor activity, bloodpressure lowering effects, bone marrow formation enhancing effects,cardiotonic activity (i.e. lowering serum cholesterol and/or increasingmyocardial metabolism and/or improving coronary artery hemodynamics),natural killer cell enhancing effects, expectorant and antitussiveproperties, immunopotentiation activities, anti-HIV activities,adrenocortical improving effects, Interleukin-1, Interleukin-2, andInterleukin-3 stimulating effects, liver-protective effects anddetoxifying effects, ionizing radiation protecting effects, anti-ulceractivity, and agents having the effect of increasing white blood cellsand hematoglobin in peripheral blood.

The above effects can be demonstrated clinically in animals includinghuman beings having been treated with a pharmaceutically effectiveamount of an extracellular agent isolated from a fungal myceliumaccording to the invention.

Method of Treatment

In one aspect the invention relates to a method of treatment of anindividual diagnosed with an immune compromised condition, said methodcomprising the steps of administering to said individual the compositionaccording to the invention or the pharmaceutical composition accordingto the invention in an amount effective in treating said immunecompromised condition.

The administered amount may in be an amount effective inprophylactically treating said immune compromised condition.

Also provided is a method of treatment of an individual recovering fromsurgery or illness and at risk of contracting an immune compromisedcondition, said method comprising the steps of administering to saidindividual the composition according to the invention or thepharmaceutical composition according to the invention in an amounteffective in boosting the immune system of said individual.

Furthermore a method of treatment of an individual diagnosed with or atrisk of contracting acquired immunodeficiency syndrome is provided, saidmethod comprising the steps of administering to said individual thecomposition according to the invention or the pharmaceutical compositionaccording to the invention in an amount effective in treating orprophylactically treating said syndrome.

The immune compromised condition may be selected from the groupconsisting of an infectious disease, a parasitic disease, haemophilusmeningitis, pneumococcal meningitis, streptococcal meningitis,staphylococcal meningitis, meningitis due to other organisms,encephalitis, viral pneumonia, pneumococcal pneumonia, other bacterialpneumonia, pneumonia due to other specified organisms except bacteria,bronchopneumonia, organism unspecific pneumonia, influenza, unspecifieddiarrhea, hepatitis unspecified, acute and subacute necrosis of theliver, chronic hepatitis, and abscess of liver.

Furthermore, the immune compromised condition may be an infectious orparasitic disease caused by, or selected from, cholera, salmonella,shigellosis, Escherichia coli, intestinal infection due to otherspecified bacteria, Clostridium difficile, viral gastroenteritis,infectious colitis, enteritis and gastroenteritis, infectious diarrhea,tuberculosis, listeriosis, pasteurellosis, mycobacterium, diphtheria,pertussis, meningococcus, streptococcus septicaemia, staphylococcussepticaemia, pneumococcal septicaemia, septicaemia due to anaerobes,septicaemia due to other gram-negative organisms, actinomycoticinfection, gas gangrene, toxic shock syndrome, necrotizing faciitis,Friedlander's bacillus, haemophilus influenzae, pseudomonas, AIDS/HIVinfections, acute poliomyelitis, Creutzfeldt-Jacob disease, subacutesclerosing panencephalitis, progressive multifocal leucoencephalopathy,unspecified slow virus infection of the central nervous system,coxsackie virus, unspecified viral meningitis, lymphocyticchoriomeningitis, unspecified viral encephalitis, chickenpox, herpeszoster, herpes simplex, viral hepatitis ‘A’, viral hepatitis ‘B’, otherspecified viral hepatitis, chronic hepatitis, abscess/acute necrosis ofliver, infectious mononucleosis, cytomegalic inclusion disease,chlamydiae, adenovirus, viral infection, syphilis, candida, unspecifiedhistoplasmosis, aspergillosis, cryptococcosis, mycoses,strongyloidiasis, intestinal parasitism, toxoplasmosis, sarcoidosis,pneumocystis carinii, post polio syndrome, haemophilus meningitis,pneumococcal meningitis, streptococcal meningitis, staphylococcalmeningitis, encephalitis, pneumonia due to adenovirus, pneumonia due torespiratory syncytial virus, pneumonia due to parainfluenza virus,pneumonia due to other virus, viral pneumonia, pneumococcal pneumonia,pneumonia due to klebsiella pneumoniae, pneumonia due to pseudomonas,pneumonia due to haemophilus influenzae, pneumonia due to streptococcus,pneumonia due to staphylococcus, and bacterial pneumonia.

The individual may be a mammal including a human being.

The pharmaceutical composition according to the invention may also beused in the manufacture of a medicament for treatment of an immunecompromised condition of an individual in need of such treatment. Theimmune compromised condition may be any of those disclosed above. Thetreatment may be prophylactic, ameliorating or curative.

The composition and the pharmaceutical compositions according to theinvention may also form part of a kit comprising said compositions and adosage regime instruction with guidelines for dose and timeadministration.

Purification and solation of Extracellular Agents

The extracellular agents such as e.g. lentinan can be isolated from theextracellular growth medium following a fermentation of e.g. Lentinusfungal mycelium, or part thereof, in a liquid growth medium.

Isolation can occur by alcohol precipitation using 70% alcohol or 96%alcohol. Suitable alcohols are C1 to C5 aliphatic alcohols, such asmethanol, ethanol, propanol, iso-propanol, n-butanol, iso-butanol,n-pentanol. The ratio between the volume of extracellular growth mediumand alcohol can vary from 5:1 to 1:5, such as 4:1, 3.1, 2:1, 1:1, 1:2,1:3, 1:4, and 1:5 depending on the concentration of alcohol and thelength of the carbon chain.

It is also possible to obtain from the precipitated fraction ofextracellular agents a fraction of such agents, includingpolysaccharides, by subjecting the alcohol precipitated extracellularagents such as polysaccharides to one or more steps selected fromdesalting, washing with an acidic solution, washing with a basicsolution, ion exchange chromatography, and gel filtration, including anycombination thereof. Desalting can be carried out using a suitablecolumn or by dialysis; suitable acidic solutions are diluted strongacids having a pH of from about 1 to about 7, such as a pH of from about2 to about 6; suitable basic solutions are diluted strong bases having apH of from about 7 to about 11, such as a pH of from about 8 to about10. Weak acids and weak bases can also be used for the washing step, ascan 70% ethanol when e.g. 96% ethanol has been used for theprecipitation.

Ion exchange chromatography and/or gel filtration are preferably usedafter desalting and/or washing of the precipitate. By using such steps,one can obtain fractions comprising extracellular agents having amolecular weight of from about 10,000 g/mol to about 20,000 g/mol,extracellular agents having a molecular weight of from about 20,000g/mol to about 40,000 g/mol, extracellular agents having a molecularweight of from about 40,000 g/mol to about 60,000 g/mol, extracellularagents having a molecular weight of from about 60,000 g/mol to about80,000 g/mol, extracellular agents having a molecular weight of fromabout 80,000 g/mol to about 100,000 g/mol, extracellular agents having amolecular weight of from about 100,000 g/mol to about 150,000 g/mol,extracellular agents having a molecular weight of from about 150,000g/mol to about 200,000 g/mol, extracellular agents having a molecularweight of from about 200,000 g/mol to about 300,000 g/mol, extracellularagents having a molecular weight of from about 300,000 g/mol to about400,000 g/mol, extracellular agents having a molecular weight of fromabout 400,000 g/mol to about 500,000 g/mol, extracellular agents havinga molecular weight of from about 500,000 g/mol to about 600,000 g/mol,extracellular agents having a molecular weight of from about 600,000g/mol to about 700,000 g/mol, extracellular agents having a molecularweight of from about 700,000 g/mol to about 800,000 g/mol, extracellularagents having a molecular weight of from about 800,000 g/mol to about900,000 g/mol, and extracellular agents having a molecular weight offrom about 900,000 g/mol to about 1,000,000 g/mol.

The purification can also involve steps actively seeking to remove e.g.proteinacious substances from e.g. a polysaccharide fraction. This canbe achieved by proteolytic degradation by e.g. proteinase K. Lipids canbe removed from a fraction by treatment with lipase or esterase.Undesirable polysaccharides can be removed by treatment with enzymessuch as glucanases, amylases, and the like.

The purity of an isolated fraction of extracellular agents can bedetermined by e.g. chromatography or spectroscopy.

One way to isolate an extracellular agent is to modify the methoddescribed by Chihara et at (1970): Fractionation and purification of thepolysaccharide with marked antitumor activity, especially lentinan fromLentinus edodes (Berk) Sing. (An edible mushroom). Cancer research, 30,2776-2781. According to the present invention, fungal biomass is removedfrom the broth with filtration and the volume of the filtrate reduced to10-20% of the original volume by evaporation. A solvent is then added tothe concentrated liquid until precipitation of the product is achieved.Solvents include alcohols such as methanol, ethanol, butanols andpropanols, as well as various ketones. The precipitate is re-suspendedand washed with alkali and acids as well as the solvent before finaldrying.

EXAMPLES

The following examples illustrate preferred embodiments of the inventionand should not be construed so as to limit the invention to theembodiments and technical results disclosed in the examples.

Example 1 Shake Flask Experiments

A medium composed of glucose, malt extract, yeast extract and peptone invarious concentrations was used for cultivating Lentinus edodes forachieving fungus growth and lentinan production in 500 ml shake flaskscontaining 200 ml of medium. The content of the various media is shownin Table 2. The flasks were shaken for 16 days at 28 degrees Centigrade.

TABLE 2 Composition of media for shake flask experiments Malt YeastPeptone Glucose Medium extract (%) extract (%) (%) (%) 1 0.3 0.3 0.5 1.02 0.3 0 0.5 1.0 3 0 0.3 0.5 1.0 4 1.0 0.3 0.5 0 5 0.3 0.3 0.5 2.0 6 0.30.3 0.5 4.0 7 0 0 0.5 1.0

All media are adjusted to pH 6

The resulting production of fungus mass and lentinan is shown in Table3:

Medium Biomass (g/l) Lentinan (mg/l) 1 2.4 42 2 0.9 15 3 1.1 14 4 0.2 05 2.3 43 6 1.8 33 7 0.3 0

The maximum amount of biomass and lentinan was produced when all mediumcomponents were present. Omission of one of the components resulted inreduced growth and lentinan production. The same thing happened if theconcentration of glucose became too high.

Example 2 Bioreactor Experiments

For these experiments, a 10-litre bioreactor with stirring equipment wasused. The reactor was sterilized and filled with 6 litres of sterilemedium. After pH adjustment to a value of about 6, the reactor wasinoculated with the content of 16 days old shake flasks. Air was addedto the reactors at a rate of 1 vvm (volume of air per volume of reactorper minute), the stirrer rotated at a rate of 200 rpm and thetemperature was kept at approx. 28 degrees Centigrade.

A. Media Compositions

The various media used are shown in Table 4.

TABLE 4 Composition of media used in bioreactor experiments Malt YeastPeptone Glucose Medium extract (%) extract (%) (%) % A 0.3 0.3 0.5 1.0 B0.3 0.3 0.5 2.0 C 0.3 0.3 1.0 1.0

The resulting concentration of biomass and lentinan achieved is shown inTable 5.

TABLE 5 Biomass and lentinan in a bioreactor with different mediaFermentation Biomass Lentinan Medium time¹ (hours) (g/l) (g/l)² A  883.97 0.17 B 240 3.62 0.23 C 211 3.48 0.10 ¹For maximum lentinanconcentration ²Containing extracellular as well as extractedintracellular lentinan.

Apparently, there is little variation in the produced amount of biomassand lentinan achieved by means of the different media. It was observed,however, that the lag time of the fermentation process was considerable.This is unproductive time and it is usual practice to reduce the lagtime by varying the amount of inoculation material used.

B. Variation of Amount of Inoculation Material Used in BioreactorExperiments.

The result of using different amounts of inoculation material is shownin Table 6:

TABLE 6 Production of biomass and lentinan in a bioreactor withdifferent amounts of inoculation material Amount of Fermentation BiomassLentinan inoculum (%) time¹ (hours) (g/l) (g/l)² 15 144 3.07 0.07 30  883.97 0.17 ¹For maximum lentinan concentration ²Contains extracellular aswell as extracted intracellular lentinan

The present results show that by changing the amount of inoculationmaterial used and the composition of the fermentation medium, it ispossible to affect the growth of Lentinus edodes and lentinanproduction. We have demonstrated that it is possible to upgrade thecultivation of L edodes from shake flasks to a bioreactor. The presentexperiments were carried out in a 10-litre reactor but there is noreason for restricting the process to the mentioned size.

The experiments show that by varying the amount of inoculation material,it is possible to affect both the lag time of the process and the amountof lentinan produced. Further experiments must be carried out forestablishing the optimal amount of inoculation material.

The experiments with respect to the composition of nutrients in thegrowth medium show that for the growth of L edodes and the production oflentinan, the medium may contain malt extract, yeast extract, peptoneand glucose. The results show that there may be a relation betweenmedium composition and production of lentinan. By increasing theconcentration of glucose to 2%, the lag time is increased but not therate of growth. This increase of the lag time also occurred in theproduction of lentinan, the production starting later at a highconcentration of glucose, but the amount of extracellular lentinanproduced was increased.

Example 3 Immunological Experiments

The result of immunological experiments with even amounts ofintracellular and extracellular material from the fermentation broth,used as described above, is presented in Table 7.

After fermentation the biomass was separated from the rest of thefermentation broth by filtration. It was then added to 1 litre ofdistilled water and this mixture was heated in an autoclave at 121° C.for two hours. The mixture was subsequently filtered and the volume ofthe filtrate was reduced by boiling to around 10% of the originalvolume. When this had cooled to room temperature, around 2 volumes ofabsolute ethanol was added to precipitate the product. The precipitatewas removed and washed with absolute ethanol, re-suspended in distilledwater and homogenised. Thereafter, 200 ml of 0.2 M cetyltrimethylammonium bromide and 0.2 M sodium hydroxide was added and the mixturewas stirred well and kept at 4° C. for 18 hours. The precipitate wasthereafter removed and washed with absolute ethanol. Then, 50 ml of a20% glacial acetic acid was added to the filtrate and the mixture wasstirred. The precipitate was subsequently removed, washed well and driedunder vacuum.

The first filtrate after the fermentation (fermentation broth less thebiomass) went though the same steps from reducing the volume by boilingonwards. Thus the only step the extracellular product did not go throughwas being kept at 121° C. for two hours.

For a determination of the immunostimulating characteristics, thefollowing method was used: 12 weeks old Sprague Dawley rats received 1mg of test compound which had been extracted from the fermentation brothin 0.5 ml 0.09 saline (i.p.) 2 days before the immunisation. Controlanimals received 1 mg casein. The animals were immunised with BSA (0.5mg) in 0.25 “Freunds Complete Adjuvant” and blood samples were obtainedafter 11 days for measurement of the antibody response. The specificanti-BSA antibody concentration was determined against an absolutestandard of antibody BSA by means of “sandwich” ELISA.

TABLE 7a Immune response (measured in anti-BSA Ig production) in ratstreated with lentinan Anti-BSA Ig Sample (μg/ml serum) Control  9Intracellular lentinan 19 Extracellular lentinan 24

TABLE 7b Immune response (measured in anti-BSA Ig production) in ratstreated with lentinan (corrected numbers) anti-BSA Ig Treatment (μg/mlserum) Control  9 Cellular lentinan 16 Ekstracellular lentinan 26

The data in Table 6b have been corrected.

The results of the immunological experiments shows that lentinan is avery active stimulator of the immune system. The extracellular productprovides a higher response than intracellular lentinan. It is,therefore, desirable to optimise the fermentation process for theproduction of extracellular lentinan. Extraction of intracellularlentinan is costly with respect to time, personnel and chemicals. It is,however, very simple to remove the extracellular product from thefermentation broth as methods for the precipitation of a polysaccharidecan be used, for example by means of various types of alcohol.

Example 4 Shake Flask Experiments

A medium composed of 15 g/l glucose, 3 g/l malt extract, 3 g/l yeastextract and 5 g/l peptone was used for cultivating Lentinus edodes toobtain fungus growth and lentinane production in 500-ml shake flaskscontaining 200 ml of medium. The flasks were shaken at 28 degreesCentigrade for a given number of days and the concentration of biomassand lentinan, respectively was measured. Typical results are given inTable 7 given below.

TABLE 7 Shake Flask Experiments Biomass Extracellular Fermentation (g/l)Product (g/l) time (days) 1.33 65 14 5.0  53 22

Extending the fermentation time led to more biomass but theconcentration of extracellular product did not show a concomitantincrease. It may be that degradation of the product became more dominantduring the extended fermentation time allowed.

Example 5 Bioreactor Experiments

The experiments were carried out in a 3-litre and a 10-litre bioreactorwith stirring equipment. The reactor was sterilized and filled with 2and 6 litres of sterile medium, respectively. Following pH adjustment toa value of about 6, the reactor was inoculated with the content of 7-8days old shake flasks. Air was supplied to the reactors and the stirringequipment performed a good mixing of the reactor content.

The temperature was kept at 28 degrees Centigrade. In the experimentsreported here, the fermentation time was 7 days unless otherwise stated.

Under these conditions, the concentration of biomass (dry weight) can beup to 3 g/l and the concentration of isolated dried polysaccharide canbe 200 mg/l. Typical results are given in Table 2.

TABLE 8 Typical Results from Bioreactor Experiment Biomass Extracellular(g/l) Product (mg/l) Comments 2.5 100 Glucose-based medium 1.6 140Sucrose-based medium

1. A method for producing an immune stimulating agent, said method comprising the steps of i) cultivating a fungus of the genus Lentinus in a liquid growth medium, wherein said cultivation results in extracellular accumulation of the immune stimulating agent, and ii) isolating the extracellularly located immune stimulating agent from the liquid growth medium.
 2. The method of claim 1, wherein the fungus belongs to the species Lentinus edodes.
 3. The method of claim 1, wherein the immune stimulating agent comprises a polysaccharide.
 4. The method of claim 1, wherein the immune stimulating agent consists of a polysaccharide.
 5. The method of claim 3, wherein the polysaccharide is a homopolymer.
 6. The method of claim 3, wherein the polysaccharide comprises D-glucose residues linked in beta-(1,3) configuration.
 7. The method of claim 3, wherein the polysaccharide comprises a backbone of beta-(1,3)-glucosyl units.
 8. The method of claim 7, wherein the polysaccharide comprises 2 glucose branches for every 5 beta-(1,3) glucosyl units in the backbone
 9. The method of claim 3, wherein the polysaccharide has a molecular weight in the range of from 400,000 g/mol to about 1,000,000 g/mol.
 10. The method of claim 1, wherein the immune stimulating agent comprises or consists of lentinan.
 11. The method of claim 1, wherein the immune stimulating agent is capable of stimulating in an individual in need of such stimulation, the production of one or more of antibodies, such as IgG, IgA, and IgE, T helper cells, interleukins, such as IL-1 and IL-2, interferon, such as IFN-gamma, natural killer cells, and macrophages.
 12. The method of any of claims 1 to 10, wherein the liquid growth medium comprises one or more typical ingredients required for growth of microbial organisms such as malt extract, yeast extract, peptone, glucose, sucrose, salts providing phosphate, magnesium and potassium, corn-steep liquor and vitamins such as thiamine, more preferably malt extract, yeast extract, peptone, and glucose.
 13. The method of any of claims 1 to 10, wherein the liquid growth medium is agitated and supplied with an oxygen source.
 14. The method of any of claims 1 to 10, wherein the growth temperature is in the range of from 23° C. to 32° C.
 15. The method of any of claims 1 to 10, wherein the fungal mycelium, and fractions thereof, are removed from the liquid growth medium prior to the isolation of the immune stimulating agent.
 16. The method of claim 15, wherein the fungal mycelium, and fractions thereof, are removed by filtration or centrifugation.
 17. The method of claim 16, wherein the immune stimulating agent is precipitated by alcohol precipitation.
 18. The method of claim 17, wherein the precipitated immune stimulating agent is further purified by washing and/or desalting.
 19. The method of claim 17, wherein the precipitated immune stimulating agent is further purified by washing and ion-exchange chromatography.
 20. The method of any of claims 18 and 19, wherein the precipitated immune stimulating agent is further purified by size exclusion chromatography or gel filtration.
 21. The method of any of claims 1 to 20, wherein the extracellularly located immune stimulating agent isolatable from the liquid growth medium is also produced intracellularly in said fungal myclium.
 22. The method of claim 21, wherein extracellularly located immune stimulating agent is immunologically distinct from intracellularly produced immune stimulating agent.
 23. An immune stimulating agent obtainable from the extracellular part of the liquid growth medium according to the method of any of claims 1 to
 22. 24. A composition comprising the immune stimulating agent according to claim 23 and a physiologically acceptable carrier.
 25. A pharmaceutical composition comprising the immune stimulating agent according to claim 23 and a pharmaceutically acceptable carrier.
 26. A method of treatment of an individual diagnosed with an immune compromised condition, said method comprising the steps of administering to said individual the composition according to claim 23 or the pharmaceutical composition according to claim 24 in an amount effective in treating said immune compromised condition.
 27. A method of treatment of an individual at risk of contracting an immune compromised condition, said method comprising the steps of administering to said individual the composition according to claim 23 or the pharmaceutical composition according to claim 24 in an amount effective in prophylactically treating said immune compromised condition.
 28. A method of treatment of an individual recovering from surgery or illness and at risk of contracting an immune compromised condition, said method comprising the steps of administering to said individual the composition according to claim 23 or the pharmaceutical composition according to claim 24 in an amount effective in boosting the immune system of said individual.
 29. A method of treatment of an individual diagnosed with or at risk of contracting acquired immunodeficiency syndrome, said method comprising the steps of administering to said individual the composition according to claim 23 or the pharmaceutical composition according to claim 24 in an amount effective in treating or prophylactically treating said syndrome.
 30. The method of any of claims 26 to 29, wherein the immune compromised condition is selected from the group consisting of an infectious disease, a parasitic disease, haemophilus meningitis, pneumococcal meningitis, streptococcal meningitis, staphylococcal meningitis, meningitis due to other organisms, encephalitis, viral pneumonia, pneumococcal pneumonia, other bacterial pneumonia, pneumonia due to other specified organisms except bacteria, bronchopneumonia, organism unspecific pneumonia, influenza, unspecified diarrhea, hepatitis unspecified, acute and subacute necrosis of the liver, chronic hepatitis, and abscess of liver.
 31. The method of any of claims 26 to 29, wherein the immune compromised condition is an infectious or parasitic disease caused by or selected from cholera, salmonella, shigellosis, Escherichia coli, intestinal infection due to other specified bacteria, Clostridium difficile, viral gastroenteritis, infectious colitis, enteritis and gastroenteritis, infectious diarrhea, tuberculosis, listeriosis, pasteurellosis, mycobacterium, diphtheria, pertussis, meningococcus, Streptococcus septicaemia, Staphylococcus septicaemia, pneumococcal septicaemia, septicaemia due to anaerobes, septicaemia due to other gram-negative organisms, actinomycotic infection, gas gangrene, toxic shock syndrome, necrotizing faciitis, Friedlander's bacillus, Haemophilus influenzae, pseudomonas, AIDS/HIV infections, acute poliomyelitis, Creutzfeldt-Jacob disease, subacute sclerosing panencephalitis, progressive multifocal leucoencephalopathy, unspecified slow virus infection of central nervous system, coxsackie virus, unspecified viral meningitis, lymphocytic choriomeningitis, unspecified viral encephalitis, chickenpox, Herpes zoster, Herpes simplex, viral hepatitis ‘A’, viral hepatitis ‘B’, other specified viral hepatitis, chronic hepatitis, abscess/acute necrosis of liver, infectious mononucleosis, cytomegalic inclusion disease, chlamydiae, adenovirus, viral infection, syphilis, Candida, unspecified histoplasmosis, aspergillosis, cryptococcosis, mycoses, strongyloidiasis, intestinal parasitism, toxoplasmosis, sarcoidosis, Pneumocystis carinii, post polio syndrome, Haemophilus meningitis, Pneumococcal meningitis, Streptococcal meningitis, Staphylococcal meningitis, encephalitis, pneumonia due to adenovirus, pneumonia due to respiratory syncytial virus, pneumonia due to parainfluenza virus, pneumonia due to other virus, viral pneumonia, pneumococcal pneumonia, pneumonia due to Klebsiella pneumoniae, pneumonia due to Pseudomonas, pneumonia due to Haemophilus influenzae, pneumonia due to Streptococcus, pneumonia due to Staphylococcus, and bacterial pneumonia.
 32. Method of any of claims 26 to 32, wherein the individual is a mammal including a human being.
 33. Use of the pharmaceutical composition according to claim 24 in the manufacture of a medicament for treatment of an immune compromised condition of an individual in need of such treatment.
 34. Use of claim 33, wherein the individual is a mammal including a human being.
 35. Use of claim 33, wherein the treatment is prophylactic, ameliorating or curative.
 36. Pharmaceutical kit comprising the composition of claim 23 or the pharmaceutical composition of claim 24 in solid form and a dosage regime instruction with guidelines for dose and times for administration. 